Abstract: In the conjugate vaccine era, Nontypeable Haemophilus influenzae (NTHi) has become the leading cause of acute otitis media (AOM), recurrent AOM, acute sinusitis and conjunctivitis in children and adults and acute exacerbations of chronic obstructive pulmonary disease (COPD) in adults. There is need to develop a vaccine against NTHi. Various vaccine candidates have been explored but still there is need for enhancement of antibody and Th17 immunity response for NTHi vaccines that will help prevent nasopharyngeal colonization and infection. In this project we will compare immunogenicity of recombinant proteins P6 and OMP26 and their fusion constructs in their lipidated and non-lipidated form in a mouse coinfection model of NTHi-AOM, we developed. Two proteins were selected based on their different function and considering P6 is naturally lipidated and native OMP26 is not but is known to induce Th17 immunity. We will test our hypothesis that lipidated protein antigens elicit higher blood and mucosal antibody levels as well as Th17 immune response (via toll-like receptor 2 activation) than non lipidated proteins. Lipidated protein antigens will elicit greater reduction in ear and nasal bacterial loads compared to nonlipidated antigens against NTHi. The contribution and mechanism of antibody mediated and TH17-mediated immunity in protection against NTHi will be compared in infant and adult mice as well as in TLR2-knock out mice using lipidated, nonlipidated and fusion constructs of vaccine candidates P6 and OMP26. We will test whether lipidated proteins produce a more robust IL-17A response from memory Th17 cells in the nasopharynx that helps traffic neutrophils and show enhanced NTHi clearance compared to nonlipidated protein antigens. Overall, the proposed studies will significantly advance our understanding of enhanced immunogenicity of recombinant proteins and molecular mechanisms underlying the lipidation regulation of TLR-2 dependent Th17- immunity in NTHi vaccine development.